Altered Bile Acid Metabolism in Vegetarians Gershon W. Hepner Bile acid kinetics were studied in a group of healthy vegetarians and a matched group of healthy control subjects. The daily fractional turnover rate of cholic acid was significantly smaller in the vegetarians than in the controls and deoxycholic acid pool size was significantly smaller in the vegetarians than in the controls. The data suggest that enterohepatic conservation of cholic acid is more efficient in vegetarians than in control subjects resulting in decreased deoxycholic acid input in vegetarian subjects. The possible significance of these data to intestinal oncogenesis is discussed.

T h e effect of diet on bile acid metabolism is incompletely understood. Dietary factors are suspected to play a role in gallstone formation in man (1-3) and in experimental animals (1). T h e prevalence of gallstones is increased when patients with hypercholesterolemia are treated with a diet high in plant sterol and low in saturated fat and cholesterol (4). Fat intake affects levels of serum cholesterol (5-12), and probably also affects fecal bile acids and neutral si:eroids (11-17). Adsorption of bile acids to various food residues may influence the rate of their loss from the intestine (18-21). Differences in bile acid metabolism due to dietai'y factors may influence stool mass and water, since some bile acids cause w a t e r secretion f r o m the intestine (22-24), and differences in the prevalence of gastrointestinal cancer in various parts of the world may also be similarly related (2527). Meat intake seems to be one of the important factors regulating serum cholesterol (5, 8-1i), From the Departme~}t of Medicine, The Milton S. Hershey Medical Cemer, The Pennsylvania State University, Hershey, Pennsylvania. This study was supported in part by Grant AM 17303 from the NIH. Address for reprint requests: (~.W. Hepner, Department of Medicine, The Milton S. Hershey Medical Center, The Pennsylvania State University, Hershey, Pennsylvania 17033.

Digestive Diseases, Vol. 20, No. 10 (October 1975)

and both fecal flora and fecal steroids are altered by a vegetarian diet (28). To assess the effect of meat on bile acid metabolism, bile acid kinetics were compared in a grouio of,vegetarians and a group of control subjects.

MATERIALS AND METHODS Subjects 10 veget.arians were studied, of whom 8 were female and 2 were male. Their ages ranged from 21-43 (28 4- 7 years, mean 4- sD) and their weight from 44-78 kg (56 4- 8 kg). The vegetarians had all excluded meat and fish, hut not eggs and milk, ft'om their diet for at least 2 yr. 10 control subjects, 7 female and 3 male, who ate meat at least 4 times per week were studied. Their ages ranged from 21-55 (34 i 7 years) and their weight from 50-72 kg (58 ::6 7 kg). A dietary history failed to disclose any significant difference in the diet of the two groups other than the meat content; fiber conteni was similar in the diet of the two groups. Bowel habit, too, did not differ between the two groups. All subjects had about one well-formed stool per day. Serum cholesterol was 154 4- 18 mg/100 ml in the vegetarians, and not dissimilar (172 • 21 mg/100 ml) in the controls. No subjects had evidence of liver disease or gallstones.

Bile Acid Kinetics A single study was performed on each subject. All studies were performed with the subjects fully ambulant and instructed to follow their usual diet. On the morning on which the siudy b~gan, each subject was given 20 ~(2i 2,4[3H]cholic acid and 10 /,Ci 24@4C]chenodeoxycho]ic acid dissolved in a beaker of water. The bile acids were obtained

935

HEPNER Table 1. Bile Acid Pool Sizes (#mol/kg) in Vegetarians and in Control Subjects Vegetarians

Controls

Subject

Cholic

Chenodeoxycholic

Deoxycholic

Total

Cholic

Chenodeoxycholic

Deoxycholic

Total

1 2 3 4 5 6 7 8 9 10

51.0 46.5 36.5 55.9 84.7 47.2 57.0 48.5 38.5 40.3

33.7 27.8 25.6 42.5 29.8 42.4 48.3 29.8 16.2 27.3

22.8 17.9 13.4 19.6 10.4 9.6 46.3 6.4 8.1 24.0

107.5 92.2 75.5 118.0 124.9 99.2 151.7 85.7 62.8 91.6

36,2 42.4 39.7 45.0 55.3 41.2 49.7 36.4 39.7 38.3

24.2 28.7 33.6 28.2 28.3 35.0 36.7 30.1 33.0 35.1

25.3 19.8 28.5 25.4 18.7 38.8 30.3 31.6 39.7 30.9

85.7 82.9 101.8 98.6 102,3 115,0 116,7 98,0 112.4 104.3

Mean 4- SE 50.6 4- 4.4 32.3 • 4,3 17.9 i 3.7* 100.9 4- 8.2 42.4 • 1.9 31.3 ~_ 1.3 28.9 • 2.1" 101.8 • 3.6 *P < 0.02 when pool size of deoxycholic acid is c o m p a r e d for the two groups.

fi'om New England Nuclear, Boston, Massachusetts, and were more than 95% pure when radiohistograms were performed on samples separated by thin-layer chromatography. On days 1, 2, 3, 4, and 7 after administration of the two radioactively labeled bile acids, a duodenal tube was passed orally. Aspiration of brown fluid 30-60 min after the passage of the tube indicated that its distal end was in the duodenum. Subjects were then given an intravenous injection of 40 units cholecystokinin (Cecekin, Stockholm, Sweden) and 2~I ml of concentrated duodenal bile were withdrawn. Samples of duodenal bile were heated to precipitate protein, filtered, and dried. The residue was redissolved in 2 ml aqueous 10% KOH and hydrolyzed for 5 hr at 15 psi and 120~ C. The tubes containing the hydrolyzed bile acids were placed in ice; 1 ml of methanol, 2 ml of 6 N HC1 and 5 ml of H20 were added. The free chotanoic acids were extracted 3 times with 5 ml of fi'esh ethyl ether, and separated by thinlayer chromatography, spotting each sample on a silicic acid plate and developing in isooctane:isopropyl ether:glacial acetic acid:butanol:water (10:5:5:3:1 vol/vol). After development of the thin-layer plates, the cholic and chenodeoxycholic acid were eluted with methanol, and an aliquot of each was added to Aquasol and radioactivity counted in a liquid scintillation counter, in which cpm were converted to dpm by external standardization. Another aliquot was assayed for bile acid mass using the steroid dehydrogenase assay (29). Bile acid kinetics were determined according to a first-order kinetics model as described by Lindstedt (30). The correlation coefficient of time against loge specific activity ranged between 0.96 and 0.99 in all studies, both in the control subjects and in the vegetarians,

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In order to determine whether there was any difference in deoxycholic acid pool size in the vegetarian diet, an aliquot of the free cholanoic acids obtained after hydrolysis of the duodenal bile acids was analyzed by gas-liquid chromatography (31). The ratio of biliary bile acids was thus determined, and the pool size of deoxycholic acid and the total bile acid pool size, calculated. Lithoeholate was less than 3% of the biliary bile acids in all specimens as determined by gas-liquid chromatography and its pool size was not calculated.

Statistics Means and standard error were determined by standard methods. Data between vegetarians and control subjects were compared using the Student's t test.

RESULTS The pool size of cholic, chenodeoxycholic, and deoxycholic acid for the two groups of subjects is shown in Table 1. The pool sizes of cholic and chenodeoxycholic acid were similar in the vegetarians and the control subjects; the pool size of deoxycholic acid was significantly smaller in the vegetarians than in the control subjects (t = 2.55, P < 0.02). The daily fractional turnover rate (DFTR) and synthesis rate of cholic acid and chenoDigestive Diseases, Vol. 20, No. 10 (October 1975)

BILE ACID METABOLISM IN VEGETARIANS Table 2. Daily Fractional Turnover Rate (DFTR) and Synthesis Rate of Cholic and Chenodeoxycholic Acid in Vegetarians and Control Subjects Vegetarians Cholic

Controls

Chenodeoxycholic

Daily Subject 1 2 3 4 5 6 7 8 9 10

Cholic

Daily

DFTR (%)

synthesis (/xmol/kg)

DFTR (%)

synthesis (#mol/kg)

DFTR (%)

24.5 17.7 25.0 14.8 13.3 28.0 45.0 5.5 20.3 21.0

12.5 7.6 9,1 8.3 11.3 13.2 25.7 2.7 7.8 8.5

13.0 12.7 12.0 11,8 18.2 24,6 30.7 11.0 13.3 16.1

4.4 3.5 3.1 5.0 5.4 10.4 14.8 3.3 2.2 4.4

29.3 33.4 38.1 27,2 17.5 36.0 18.5 47.1 49.3 41.2

Mean + sE 2 1 . 5 •

10.7 ~- 1.9 1 6 . 3 •

5.7 • 1.3 3 3 . 0 •

Chenodeoxycholic

Daily synthesis

Daily

(/xmol/kg)

DFTR (%)

synthesis (/zmol/kg)

10.6 14.2 15.1 12.2 9.7 14.8 9.2 17.1 19.6 15.8

18.2 17.3 27.3 20.0 11.1 17.2 16.1 33.0 41.0 29.3

4.4 5.0 9.2 5.6 3.1 6.0 5.9 9.9 13.5 10.3

1 3 , 8 t 1.1 23.1 •

7.3!1.0

*P < 0.02 when DFTR of cholic acid is compared for the two groups.

deoxycholic acid is shown for the two groups of subjects in Table 2. The D F T R of cholic acid was significantly less (t = 2.56, P < 0.02) in the vegetarians than in the controls; the D F T R of chenodeoxycholic acid was similar in the two groups of subjects. There was no difference in the synthesis rate of the primary bile acids in the two groups.

DISCUSSION

The data from this study indicate that the D F T R of cholic acid is significantly less in vegetarians than in control subjects. The D F T R of cholic acid is determined by three factors: (1) efficiency of ileal reabsorption; (2) bacterial dehydroxylation, which, if it occurs proximal to a site where bile acids are reabsorbed, may result in conservation of the steroid moiety of the bile acid as its bacterial metabolite deoxycholic acid; and (3) the rate of enterohepatic recycling, which determines the number of times per day the bile acid pool is exposed to possible nonDigestive Diseases, Vol. 20, No. 10 (October 1975)

r e a b s o r p t i o n by the intestine and dehydroxylation. It seems unlikely that mucosal function is altered in vegetarian subjects. Luminal contents are more likely to be altered and could theoretically affect bile acid kinetics in three ways. First, they may affect intestinal transit time. Although this was apparently similar between the two groups of subjects, since bowel habit was similar, it is possible that differences in transit time not clinically apparent nevertheless occurred. Transit time affects the conversion of cholic acid to deoxycholic acid (32, 33), as does dietary fiber (34); hence increased transit time in the vegetarians may have decreased their cholic acid fractional turnover rate by decreasing the conversion of cholic to deoxycholic acid. Secondly, certain luminal contents may adsorb bile acids (17-21) and sequester them from the enterohepatic circulation. While this may have occurred in some of the vegetarians we studied, it would not explain our data since such sequestration would tend to decrease enterohepat937

HEPNER

ic conservation of bile acids and thus increase, rather than decrease, their fractional turnover rate. A third factor that may be modified by luminal contents is bacterial flora. Fecal flora in a group of English vegetarians contained fewer Bacteroides capable of dehydroxylation than were found in a control group (28), and a similar observation has been made in a group of Asian vegetarians (25). If such a phenomenon occurred in the vegetarians in this study, it would also have contributed to the decreased fractional turnover rate of cholic acid. It is theoretically possible that the vegetarian diet led to a decreased fractional turnover rate of cholic acid by decreasing the enterohepatic recycling rate. There is no evidence that vegetable protein and fat are less effective than animal protein and fat in stimulating the endogenous release of cholecystokinin-pancreozymin (CCK-PZ), and this possibility merits further investigation.

decreased deoxycholic acid pool, input of deoxycholic acid into the enterohepatic circulation would be normal if deoxycholate D F T R were increased. Although such studies might best be performed with 24-14C-labeled bile acids because of problems arising out of the use of 3Hlabeled bile acids (43), LaRusso et al have indicated that bile acid kinetics using the latter are acceptably valid, the error in their use being small (44). ACKNOWLEDGMENT

The invaluable assistance of Mrs. Judith Marks is gratefully acknowledged. REFERENCES

The study indicates that meat may have an important effect on bile acid metabolism. The decreased deoxycholic acid pool size in the vegetarians may have important implications in connection with the prevalence of bowel cancer. This is recognized as being less common in developing countries than in developed countries (35), and it has been suggested that the diet consumed in these countries may cause decreased deoxycholic acid or other secondary bile acids that may be oncogenic (36-39) to reach the left side of the colon. Since meat consumption is also less in most developing countries than in the developed countries, it is possible that the presence of meat in the diet, by increasing deoxycholic acid pool size and input into the enterohepatic circulation, may lead to such an oncogenic effect (40, 41). Previous efforts to link deoxycholic acid input and the diet in different areas of the world have laid greater emphasis on dietary fiber content (35, 42), but this study suggests that the effect of meat p e r se requires further investigation. Furthermore, deoxycholic acid kinetics in vegetarians should be determined, since even in the presence of a

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BII.E ACID METABOLISM iN VEGETARIANS

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Digestive Diseases, Vol. 20, No. 10 (October 1975)

Altered bile acid metabolism in vegetarians.

Bile acid kinetics were studied in a group of healthy vegetarians and a matched group of healthy control subjects. The daily fractional turnover rate ...
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